Purification of cholera toxin and its subunits: new methods of preparation and the use of hypertoxinogenic mutants

Abstract

Cholera toxin was obtained in pure form by fractionation on two phosphocellulose columns successively. Cholera toxin and choleragenoid were quantitatively and selectively adsorbed to the first column in 10 mM phosphate buffer, pH 7.0, and were subsequently eluted with buffer of high ionic strength. The toxin was then separated from choleragenoid on the second column by chromatography at pH 8.3. The toxin obtained was highly active and pure as judged by electrophoresis, isoelectric focusing, and various immunological and chemical tests. Pure choleragenoid was a by-product of the procedure. The A1 chain of the toxin was obtained in pure form by treating phosphocellulose-bound toxin with urea and a reducing agent. The anionic Al peptide was thereby released, leaving a complex of the B and A2 chains (A25B) bound to the resin. The latter was then eluted and further purified to obtain nontoxic antigen. The overall yields of cholera toxin and choleragenoid were increased twoto threefold by the use of hypertoxinogenic mutants of Vibrio cholerae.

Genetic mapping of toxin regulatory mutations in Vibrio cholerae

“…This behavior was consistent with the apparent coordinate control of A and B subunit synthesis previously observed for various hypotoxinogenic mutants (6,22). These observations suggested that hypertoxinogenic mutations, as well as all tox mutations (6,10,16,22) thus far described, belong to the broad class of "regulatory" mutations which alter the levels of toxin produced rather than the toxin molecule per se.…”

“…Mutants altered in the production of toxin (tox mutants) have been isolated in several laboratories, but most of these appear to be regulatory mutants that produce greatly reduced levels of toxin (6,10,16,22). A tox regulatory site that mediates the hypoproduction of cholera toxin was reported by Vasil and co-workers (3,23) to be weakly linked to the his-1 locus of V. cholerae.…”

“…Several presumably allelic mutations were also mapped in the same region of the chromosome, but the nature of these mutations was not determined. More recently, Mekalanos et al (15,16) isolated mutants that produced elevated levels of cholera toxin compared to the wild-type parent strain. These hypertoxinogenic (Htx) mutants appeared to produce equivalently elevated amounts of both the A and B chains of the toxin.…”

Role of cyclic adenosine monophosphate in amylase release from dissociated rat pancreatic acini

“…The following were purchased: Male Sprague Dawley rats from ARS/Sprague Dawley and Holtzman Co., Madison, WI, U.S.A.; chromatographically purified collagenase from Millipore, Freehold, NJ, U.S.A.; a-Chymotrypsin from Worthington Biochemical Corp., Freehold, NJ, U.S.A.; hyaluronidase, bovine plasma albumin (fraction V), soybean trypsin inhibitor, L-glutamine, fl-nicotinamide adenine dinucleotide (DPN), cholera toxin (vibrio cholera, lyophilized powder prepared and purified according to Mekalanos, Collier & Romig (1978)…”

Proteine der Zellwand von Escherichia coli: Eine dem thermolabilen Enterotoxin (LT) ähnliche Substanz

“…Nach Zentrifugation 30 min bei 10000 X g wurde der Uberstand fur weitere Untersuchungen verwendet (KUNREL u. ROBERTSOX 1979 b). -Hexametaphosphat : 0,25 g pro 100 ml Extrakt, dann Zutropfen von verdiinnter Essigsiiure bi8 pH 4,7 (MEKALANOS et al 1978).…”